Cycloheximide (chx)

All chemicals were obtained from Nacalai Tesque (Kyoto, Japan), Wako (Osaka, Japan), Corning (Corning, NY, USA), Qiagen (Hilden, Germany), Invitrogen (Carlsbad, CA, USA), and Sigma-Aldrich (St. Louis, MO, USA). Tunicamycin, U0126, SP600125, and cycloheximide were purchased from Nacalai Tesque (Kyoto, Japan). GW9662 (Sigma-Aldrich, St. Louis, MO, USA), rosiglitazone (LKT Laboratories, St. Paul, MN, USA), and PBA (Santa Cruz Biotechnology, Dallas, TX, USA) were acquired from the indicated companies.

Fucoxanthinol was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Cycloheximide and Phosphatase Inhibitor Cocktail (EDTA free) were purchased from Nacalai Tesque (Kyoto, Japan). MG-132 was purchased from Peptide Institute (Osaka, Japan).

Recombinant human IGF-I was kindly donated by T Ohkuma (Astellas Pharma Inc., Tokyo, Japan). Recombinant human EGF was purchased from Thermo Fisher. Prior to ligand stimulation, the cells were serum-starved for 12 hr in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 0.1% bovine serum albumin (BSA), and then treated with the ligand (100 nM IGF-I or 100 nM EGF) for the indicated time. When needed, cells were preincubated for 30 min with chemical inhibitors at the following concentrations: 250 μg/ml leupeptin (PEPTIDE INSTITUTE, INC., Osaka, Japan), 10 μg/ml pepstatin A (Sigma-Aldrich), 100 nM Torin1 (Cayman Chemical), 100 nM rapamycin (Sigma-Aldrich), 0.1 mM primaquine (Sigma-Aldrich), and 10 μg/ml cycloheximide (Nacalai Tesque, Inc., Kyoto, Japan).
After the treatment, the extraction of cell lysate and immunoblotting were performed as described previously (Yoneyama et al., 2013 (link)). Densitometry was performed in the linear phase of the exposure by using ImageJ software. The results were expressed as the percent of max, which corresponds to the highest value of phosphorylation among the time course experiments of control cells. Values represent means ±SEM from at least three independent experiments.

Human recombinant TGF-β1 was obtained from R&D Systems. Cycloheximide was purchased from Nacalai Tesque. SB431542 was obtained from Calbiochem. LY29400 was obtained from Sigma-Aldrich. Y27632 was purchased from FUJIFILM Wako Pure Chemical Co. Rhodamine-phalloidin was purchased from Cytoskeleton, Inc. The following antibodies were also used: anti-Smad2/3 (610843, BD Bioscience); anti-phospho-Smad1 (9511, Cell Signaling Technology); anti-phosho-Smad2 (138D4, Cell Signaling Technology); anti-phosho-Smad3 (9520, Cell Signaling Technology); anti-Smad4 (B-8, Santa Cruz Biotechnology); anti-E-cadherin (610182, BD Bioscience); anti-FLAG (M2; Sigma-Aldrich); anti-Rac1 (23A8; Millipore); anti-phospho-Akt (9271, Cell Signaling Technology); and anti-α-tubulin (DM1A) (T9026, Sigma-Aldrich). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (115-035-003, Jackson Immuno Research) and goat anti-rabbit IgG (111-035-003, Jackson Immuno Research) were used as secondary antibodies.

MEFs were prepared as described previously^{32 (link)}. The HEK293T cell line and wild-type and p53^−/−MEFs were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS). The following reagents were used for treating cells: cycloheximide (Cat# 06741-04; Nacalai Tesque, Inc., Kyoto, Japan), MG132 (Cat# 474790; Merck KGaA, Darmstadt, Germany), lipopolysaccharide (Cat# L2637; Merck KGaA), TNF-α (Cat# 300-01A; PeproTech, Inc., Rocky Hill, NJ, USA), IL-1β (Cat# 200-01B; PeproTech, Inc.), dichloroacetate (Cat# 347795; Merck KGaA), and 2-deoxy-D-glucose (Cat# D8375; Merck KGaA).

HeLa4 (link) and HepG2 (a gift from Dr. H. Sumimoto. Kyushu Univ.) were cultured in DMEM supplemented with 10% FBS (Biowest) in 5% CO₂ and 95% air4 (link). Chinese hamster ovary (CHO)-K1and adaps ZPEG2512 (link) were cultured in Ham’s F-12 medium supplemented with 10% FBS in 5% CO₂ and 95% air2 (link). Cells were cultured in the presence of several types of inhibitors as follows: CHO-K1 cells were cultured in the presence of 80 μM Dynasore (Sigma)23 (link), 25 μM CPZ (Sigma), or 30 μM MiTMAB (TOCRIS) for 5 h. ZPEG251 was treated with 10 μM PP2 (Sigma) for 5 h in the presence of purified plasmalogens4 (link). CHO-K1 cells were treated with 25 μg/ml nystatin (Sigma) for 1 h and further cultured in the presence of 10 μg/ml cycloheximide (Nacalai Tesque) for the indicated period. Knockdown of FLOT1, CAV1, and CDC50A was performed by transfecting dsRNAs against respective genes to HeLa cells and the cells were cultured for 3 days. The target sequences of the dsRNA are as follows: human Flotillin-1–68, 5′-GGGCATCAGTGTGGTTAGCTACACT-3′; human Flotillin-1–69, 5′- CGGGAAGCTAAAGCCAAGCAGGAAA-3′ (Invitrogen); human Caveolin1-9504, 5′-CCCTAAACACCTCAACGATTT-3′ (Sigma); human CDC50A-22, 5′-GAGCTATTGCCAACAGCATTT-3′; and human CDC50A-23, 5′-CGTGTTTATGTATTATGGATT-3′ (Sigma).

Insulin, 3-isobutyl-1-methylxanthine (IBMX), and dexamethasone were purchased from CAYMAN CHEMICAL COMPANY. Dimethyl sulfoxide (DMSO) and cycloheximide (CHX) were purchased from Nacalai Tesque (Tokyo, Japan). MG132 was purchased from Calbiochem (San Diego, CA, USA). An anti-phospho-MEK1/2 antibody (S217/221), anti-MEK1/2 antibody, anti-phospho-ERK1/2 antibody (T202/Y204), anti-ERK1/2 antibody, anti-phospho-Akt antibody (S473), anti-Akt antibody, anti-phospho-Insulin receptor β (IRβ) antibody (Y1146), anti-IRβ antibody, anti-phospho C/EBPβ antibody (T235), anti-Insulin receptor substrate 1 (IRS1) antibody, and anti-IRS2 antibody were purchased from Cell Signaling Technology (Danvers, MA, USA). An anti-C/EBPδ antibody, anti-C/EBPβ antibody, anti-C/EBPα antibody, anti-PPARγ antibody, and anti-β-actin antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).